A single ubiquitin is sufficient for cargo protein entry into MVBs in the absence of ESCRT ubiquitination
Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52246, USA.The Journal of Cell Biology (Impact Factor: 9.83). 01/2011; 192(2):229-42. DOI: 10.1083/jcb.201008121
ESCRTs (endosomal sorting complexes required for transport) bind and sequester ubiquitinated membrane proteins and usher them into multivesicular bodies (MVBs). As Ubiquitin (Ub)-binding proteins, ESCRTs themselves become ubiquitinated. However, it is unclear whether this regulates a critical aspect of their function or is a nonspecific consequence of their association with the Ub system. We investigated whether ubiquitination of the ESCRTs was required for their ability to sort cargo into the MVB lumen. Although we found that Rsp5 was the main Ub ligase responsible for ubiquitination of ESCRT-0, elimination of Rsp5 or elimination of the ubiquitinatable lysines within ESCRT-0 did not affect MVB sorting. Moreover, by fusing the catalytic domain of deubiquitinating peptidases onto ESCRTs, we could block ESCRT ubiquitination and the sorting of proteins that undergo Rsp5-dependent ubiquitination. Yet, proteins fused to a single Ub moiety were efficiently delivered to the MVB lumen, which strongly indicates that a single Ub is sufficient in sorting MVBs in the absence of ESCRT ubiquitination.
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- "We therefore examined whether ubiquitination plays a role in the NVT pathway by using a DUb (deubiquitinating enzyme) fusion approach (Stringer and Piper, 2011). Similar to the observations in S. cerevisiae (Stringer and Piper, 2011), fusing a DUb, but not its enzymatically inactive form, to MVB pathway cargos such as Ncs2 prevented the transport into vacuoles (Figure S4H). When Nbr1 was fused to either one of two DUbs, its vacuolar targeting was abolished, whereas Nbr1 fused to the catalytically inactive forms of these DUbs still accumulated inside vacuoles (Figures 4E and 4F). "
ABSTRACT: Autophagy transports cytosolic materials into lysosomes/vacuoles either in bulk or selectively. Selective autophagy requires cargo receptor proteins, which usually link cargos to the macroautophagy machinery composed of core autophagy-related (Atg) proteins. Here, we show that fission yeast Nbr1, a homolog of mammalian autophagy receptor NBR1, interacts with and facilitates the transport of two cytosolic hydrolases into vacuoles, in a way reminiscent of the budding yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy. We term this pathway Nbr1-mediated vacuolar targeting (NVT). Surprisingly, unlike the Cvt pathway, the NVT pathway does not require core Atg proteins. Instead, it depends on the endosomal sorting complexes required for transport (ESCRTs). NVT components colocalize with ESCRTs at multivesicular bodies (MVBs) and rely on ubiquitination for their transport. Our findings demonstrate the ability of ESCRTs to mediate highly selective autophagy of soluble cargos, and suggest an unexpected mechanistic versatility of autophagy receptors.
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- "ILV formation and cargo sorting are mediated by the endosomal sorting complex required for transport (ESCRT) machinery (Raiborg and Stenmark, 2009). Previous studies indicate that K63-linked polyubiquitylation of cargo proteins specifically functions as an ESCRT-dependent sorting signal to ILVs rather than as an internalization signal (Huang et al., 2006; Barriere et al., 2007; Lauwers et al., 2010; Erpapazoglou et al., 2012), although other reports suggest that multiple or even single monoubiquitylation is sufficient for MVB sorting (Stringer and Piper, 2011). Thus, the exact role of K63- linked ubiquitylation in endocytic processes remains controversial. "
ABSTRACT: In Caenorhabditis elegans, fertilization triggers endocytosis and rapid turnover of maternal surface membrane proteins in lysosomes, although the precise mechanism of this inducible endocytosis is unknown. We found that high levels of K63-linked ubiquitin chains transiently accumulated on endosomes upon fertilization. Endocytosis and the endosomal accumulation of ubiquitin were both regulated downstream of the anaphase-promoting complex, which drives the oocyte's meiotic cell cycle after fertilization. The clearance of maternal membrane proteins and the accumulation of K63-linked ubiquitin on endosomes depended on UBC-13 and UEV-1, which function as an E2 complex that specifically mediates chain elongation of K63-linked polyubiquitin. CAV-1-GFP, an endocytic cargo protein, was modified with K63-linked polyubiquitin in a UBC-13/UEV-1-dependent manner. In ubc-13 or uev-1 mutants, CAV-1-GFP and other membrane proteins were internalized from the plasma membrane normally after fertilization. However, they were not efficiently targeted to the multivesicular body (MVB) pathway but recycled to the cell surface. Our results suggest that UBC-13-dependent K63-linked ubiquitylation is required for proper MVB sorting rather than for internalization. These results also demonstrate a developmentally controlled function of K63-linked ubiquitylation.
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